The rat cultured pineal gland was set up in order to study tetrahydrobiopterin (BH4) regulation and turnover. When pineals from 250 g rats were cultured, pineal BH4 decreased to 50% of control levels after 24 hours of culture. This was due to release of BH4, detected as biopterin, into the culture medium. A suitable culturesystem was obtained when better tissue oxygenation was achieved by using smaller pineals from 100 g rats. Under these conditions pineal BH remained stable for 24 hours in culture, released into the culture medium. Pineal levels of the cellular marker, with an increase at 48 hours to 150% of initial levels, and less BH4 was lactate dehydrogenase (LDH) were constant during culture, indicating that most of the pineal cells remained viable. The ratio of reduced to oxidized biopterin did not change during culture, suggesting that BH4 regeneration from guinonoid dihydrobiopterin (q-BH2) was occurring normally in the cultured glands. In contrast, cultured murine p815 mastocytoma cells were not a suitable system since BH4 levels fluctuated and cells appeared to multiply during culture. A biopterin-bovine serum albumin conjugate is being used as an immunogen in rabbits to develop antibodies to biopterin, ultimately required for the development of an immunoassay for biopterin.